Structural basis for a degenerate tRNA identity code and the evolution of bimodal specificity in human mitochondrial tRNA recognition

Animal mitochondrial gene expression relies on specific interactions between nuclear-encoded aminoacyl-tRNA synthetases and mitochondria-encoded tRNAs. Their evolution involves an antagonistic interplay between strong mutation pressure on mtRNAs and selection pressure to maintain their essential function. To understand the molecular consequences of this interplay, we analyze the human mitochondrial serylation system, in which one synthetase charges two highly divergent mtRNASer isoacceptors. We present the cryo-EM structure of human mSerRS in complex with mtRNASer(UGA), and perform a structural and functional comparison with the mSerRS-mtRNASer(GCU) complex. We find that despite their common function, mtRNASer(UGA) and mtRNASer(GCU) show no constrain to converge on shared structural or sequence identity motifs for recognition by mSerRS. Instead, mSerRS evolved a bimodal readout mechanism, whereby a single protein surface recognizes degenerate identity features specific to each mtRNASer. Our results show how the mutational erosion of mtRNAs drove a remarkable innovation of intermolecular specificity rules, with multiple evolutionary pathways leading to functionally equivalent outcomes.

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Bernhard Kuhle
Jul 11, 2023 Cryo-EM data collection was automated using the Leginon data collection software (Suloway et et al. J Struct Biol, 2005). Data Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A description of any restrictions on data availability -For clinical datasets or third party data, please ensure that the statement adheres to our policy Research involving human participants, their data, or biological material Policy information about studies with human participants or human data. See also policy information about sex, gender (identity/presentation), and sexual orientation and race, ethnicity and racism.

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Replication Randomization Blinding Reporting for specific materials, systems and methods All data generated or analyzed during this study are included in this published article (and its supplementary information files). The cryo-EM map of mSerRS-mtRNASer(UGA) has been deposited in the Electron Microscopy Data Bank (EMDB) under the accession code EMD-29070. Atomic coordinates of the model have been deposited in the Protein Data Bank (PDB) under accession code 8FFY. The atomic coordinates used for molecular replacement or structural comparison were downloaded from the PDB: 1SER, 3L0U, 4TRA, 5UD5, 6YDP, 6ZM6, 7ONU, 7TZB, 7U2A, 7U2B. All tRNA gene sequences were retrieved from the tRNAdb/mitotRNAdb (http://trna.bioinf.uni-leipzig.de/), genomic tRNA database (GtRNAdb; http://gtrnadb.ucsc.edu/) or the National Center for Biotechnology Information database (NCBI; NC027264). Source data are provided with this paper.
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Sample sizes were not predetermined using statistical methods. For cryo-EM structure determination, a total of 3448 movies were collected for the tRNASer(UGA) dataset. The resulting particle stacks contained 2.7M particles. For cryo-EM analysis, the sample size was governed by the quality of the resulting cryo-EM map and was considered adequate when a sufficiently high quality of the cryo-EM map was obtained to perform structural fitting and atomic model building. The in vitro kinetic experiments were repeated three times to demonstrate reproducibility and to allow appropriate estimates of error by statistical analysis.
In cryo-EM analysis, micrographs with ice were excluded. Particles belonging to 2D/3D classes that did not contain high-resolution structural features were discarded and not used in the final 3D reconstruction. No data were excluded from biochemical analyses.
Cryo-EM analysis was performed once, with 2.7M particles picked from a total of 3448 movies. Structural refinement was performed for multiple rounds with distinct sets of particles, resulting in the same density maps. In vitro biochemical experiments were repeated three times with consistent results. Purification of wild-type mSerRS was repeated two times (n=2), yielding similar and reproducible aminoacylation activities from each preparation.
This study did not include treatment groups.
Due to the nature of the project (understand molecular function of human mitochondrial aaRS/tRNA systems), investigators were not blinded and were aware of the sequence characteristics of all proteins/tRNAs used in the reported experiments.

April 2023
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